Journal of Cell Science

Rabbits' feet and four-leaf clovers II. By Caveman.

Publication Date: 2010 Sep 1 PMID: 20803784
Authors:
Journal: J Cell Sci

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Spatially defined Dsh-Lgl interaction contributes to directional tissue morphogenesis.

Publication Date: 2010 Sep 15 PMID: 20736316
Authors: Kaplan, N. A. - Tolwinski, N. S.
Journal: J Cell Sci

The process of epithelial morphogenesis defines the structure of epidermal tissue sheets. One such sheet, the ventral epidermis of the Drosophila embryo, shows both intricate segmental patterning and complex cell organization. Within a segment, cells produce hair-like denticles in a stereotypical and highly organized pattern over the surface of the tissue. To understand the cell biological basis of this process, we examined cell shapes and alignments, and looked for molecules that showed an asymmetric distribution in this tissue. We found that apical polarity determinants and adherens junctions were enriched at the dorsal and ventral borders of cells, whereas basolateral determinants were enriched at the anterior and posterior borders. We report that the basolateral determinant Lgl has a novel function in the planar organization of the embryonic epidermis, and this function depends on Dsh and myosin. We conclude that apical-basal proteins, used to establish polarity within a cell, can be independently co-opted to function in epithelial morphogenesis.

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Specific replication origins promote DNA amplification in fission yeast.

Publication Date: 2010 Sep 15 PMID: 20736315
Authors: Kiang, L. - Heichinger, C. - Watt, S. - Bahler, J. - Nurse, P.
Journal: J Cell Sci

To ensure equal replication of the genome in every eukaryotic cell cycle, replication origins fire only once each S phase and do not fire after passive replication. Failure in these controls can lead to local amplification, contributing to genome instability and the development of cancer. To identify features of replication origins important for such amplification, we have investigated origin firing and local genome amplification in the presence of excess helicase loaders Cdc18 and Cdt1 in fission yeast. We find that S phase controls are attenuated and coordination of origin firing is lost, resulting in local amplification. Specific origins are necessary for amplification but act only within a permissive chromosomal context. Origins associated with amplification are highly AT-rich, fire efficiently and early during mitotic S phase, and are located in large intergenic regions. We propose that these features predispose replication origins to re-fire within a single S phase, or to remain active after passive replication.

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Exocytosis, dependent on Ca2+ release from Ca2+ stores, is regulated by Ca2+ microdomains.

Publication Date: 2010 Sep 15 PMID: 20736314
Authors: Low, J. T. - Shukla, A. - Behrendorff, N. - Thorn, P.
Journal: J Cell Sci

The relationship between the cellular Ca(2+) signal and secretory vesicle fusion (exocytosis) is a key determinant of the regulation of the kinetics and magnitude of the secretory response. Here, we have investigated secretion in cells where the exocytic response is controlled by Ca(2+) release from intracellular Ca(2+) stores. Using live-cell two-photon microscopy that simultaneously records Ca(2+) signals and exocytic responses, we provide evidence that secretion is controlled by changes in Ca(2+) concentration [Ca(2+)] in relatively large-volume microdomains. Our evidence includes: (1) long latencies (>2 seconds) between the rise in [Ca(2+)] and exocytosis, (2) observation of exocytosis all along the lumen and not clustered around Ca(2+) release hot-spots, (3) high affinity (K(d) =1.75 muM) Ca(2+) dependence of exocytosis, (4) significant reduction in exocytosis in the prescence of cytosolic EGTA, (5) spatial exclusion of secretory granules from the cell membrane by the endoplasmic reticulum, and (6) inability of local Ca(2+) responses to trigger exocytosis. These results strongly indicate that the control of exocytosis, triggered by Ca(2+) release from stores, is through the regulation of cytosolic [Ca(2+)] within a microdomain.

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Growth differentiation factor 9 signaling requires ERK1/2 activity in mouse granulosa and cumulus cells.

Publication Date: 2010 Sep 15 PMID: 20736313
Authors: Sasseville, M. - Ritter, L. J. - Nguyen, T. M. - Liu, F. - Mottershead, D. G. - Russell, D. L. - Gilchrist, R. B.
Journal: J Cell Sci

Ovarian folliculogenesis is driven by the combined action of endocrine cues and paracrine factors. The oocyte secretes powerful mitogens, such as growth differentiation factor 9 (GDF9), that regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation. This study investigated the role of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase 1 and 2 (ERK1/2; also known as MAPK3/1) signaling pathway on GDF9 action on granulosa cells. Results show that mitogenic action of the oocyte is prevented by pharmacological inhibition of the EGFR-ERK1/2 pathway. Importantly, EGFR-ERK1/2 activity as well as rous sarcoma oncogene family kinases (SFK) are required for signaling through SMADs, mediating GDF9, activin A and TGFbeta1 mitogenic action in granulosa cells. GDF9 could not activate ERK1/2 or affect EGF-stimulated ERK1/2 in granulosa cells. However, induction of the SMAD3-specific CAGA reporter by GDF9 in granulosa cells required active EGFR, SFKs and ERK1/2 as did GDF9-responsive gene expression. Finally, the EGFR-SFKs-ERK1/2 pathway was shown to be required for the maintenance of phosphorylation of the SMAD3 linker region. Together our results suggest that receptivity of granulosa cells to oocyte-secreted factors, including GDF9, is regulated by the level of activation of the EGFR and resulting ERK1/2 activity, through the requisite permissive phosphorylation of SMAD3 in the linker region. Our results indicate that oocyte-secreted TGFbeta-like ligands and EGFR-ERK1/2 signaling are cooperatively required for the unique granulosa cell response to the signal from oocytes mediating granulosa cell survival and proliferation and hence the promotion of follicle growth and ovulation.

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Isolation of a small vasohibin-binding protein (SVBP) and its role in vasohibin secretion.

Publication Date: 2010 Sep 15 PMID: 20736312
Authors: Suzuki, Y. - Kobayashi, M. - Miyashita, H. - Ohta, H. - Sonoda, H. - Sato, Y.
Journal: J Cell Sci

Upon stimulation with angiogenic factors, vascular endothelial cells (ECs) secrete a negative-feedback regulator of angiogenesis, vasohibin-1 (VASH1). Because VASH1 lacks a classical signal sequence, it is not clear how ECs secrete VASH1. We isolated a small vasohibin-binding protein (SVBP) composed of 66 amino acids. The level of Svbp mRNA was relatively high in the bone marrow, spleen and testes of mice. In cultured ECs, Vash1 mRNA was induced by VEGF, and Svbp mRNA was expressed constitutively. The interaction between VASH1 and SVBP was confirmed using the BIAcore system and immunoprecipitation analysis. Immunocytochemical analysis revealed that SVBP colocalized with VASH1 in ECs. In polarized epithelial cells, SVBP accumulated on the apical side, whereas VASH1 was present throughout the cells and partially colocalized with SVBP. Transfection of SVBP enhanced VASH1 secretion, whereas knockdown of endogenous SVBP markedly reduced VASH1 secretion. SVBP increased the solubility of VASH1 protein in detergent solution and inhibited the ubiquitylation of VASH1 protein. Moreover, co-transfection of SVBP significantly augmented the inhibitory effect of VASH1 on EC migration. These results indicate that SVBP acts as a secretory chaperone for VASH1 and contributes to the anti-angiogenic activity of VASH1.

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Multisite phosphorylation of Erk5 in mitosis.

Publication Date: 2010 Sep 15 PMID: 20736311
Authors: Diaz-Rodriguez, E. - Pandiella, A.
Journal: J Cell Sci

The MAP kinase Erk5 plays important roles in cellular proliferation, and has recently been implicated in the regulation of mitosis. The classic pathway of Erk5 activation involves dual phosphorylation at its TEY microdomain by the upstream regulating kinase MEK5. Here we describe a second pathway that controls Erk5 phosphorylation. This pathway is activated in mitotic cells and involves kinase activities distinct from MEK5. Studies aimed at identifying these kinases suggested that CDK1 activity is required to sustain Erk5 phosphorylation in mitosis, as treatment with RO3306, a CDK1 inhibitor, reversed mitotic phosphorylation of Erk5. Moreover, CDK1 co-precipitated with Erk5 in mitotic cells. The mitotic phosphorylation of Erk5 occurs at multiple sites located at its unique C-terminal region, within an Erk5 subdomain that has formerly been implicated in the control of the subcellular location of Erk5. Furthermore, molecular studies indicated that phosphorylation at these sites may participate in the control of the transit of Erk5 between the cytosol and the nucleus, in addition to regulating its transcriptional activity. Together, our results demonstrate the existence of a second Erk5 phosphorylation pathway, that is activated in mitosis, and that may participate in the regulation of Erk5 functions.

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Heteromultimeric TRPML channel assemblies play a crucial role in the regulation of cell viability models and starvation-induced autophagy.

Publication Date: 2010 Sep 15 PMID: 20736310
Authors: Zeevi, D. A. - Lev, S. - Frumkin, A. - Minke, B. - Bach, G.
Journal: J Cell Sci

The mucolipin (TRPML) subfamily of transient receptor potential (TRP) cation channels consists of three members that play various roles in the regulation of membrane and protein sorting along endo-lysosomal pathways. Loss-of-function mutations in TRPML1 cause the neurodegenerative lysosomal storage disorder, mucolipidosis type IV (MLIV), whereas a gain-of-function mutation in TRPML3 is principally implicated in the hearing-impaired and abnormally pigmented varitint-waddler mouse. Currently, TRPML2 is not implicated in any pathological disorder, but we have recently shown that it is a functional cation channel that physically interacts with TRPML1 and TRPML3 to potentially regulate lysosomal integrity. Here, we show that mutant TRPMLs heteromultimerize with other mutant and wild-type TRPMLs to regulate cell viability and starvation-induced autophagy, a process that mediates macromolecular and organellar turnover under cell starvation conditions. Heteromultimerization of dominant-negative TRPMLs with constitutively active TRPMLs rescues cells from the cytotoxic effects of TRPML constitutive activity. Moreover, dominant-negative TRPML1 channels, including a mutant channel directly implicated in MLIV pathology, also inhibit starvation-induced autophagy by interacting with and affecting native TRPML channel function. Collectively, our results indicate that heteromultimerization of TRPML channels plays a role in various TRPML-regulated mechanisms.

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The myotubularin phosphatase MTMR4 regulates sorting from early endosomes.

Publication Date: 2010 Sep 15 PMID: 20736309
Authors: Naughtin, M. J. - Sheffield, D. A. - Rahman, P. - Hughes, W. E. - Gurung, R. - Stow, J. L. - Nandurkar, H. H. - Dyson, J. M. - Mitchell, C. A.
Journal: J Cell Sci

Phosphatidylinositol 3-phosphate [PtdIns(3)P] regulates endocytic trafficking and the sorting of receptors through early endosomes, including the rapid recycling of transferrin (Tfn). However, the phosphoinositide phosphatase that selectively opposes this function is unknown. The myotubularins are a family of eight catalytically active and six inactive enzymes that hydrolyse PtdIns(3)P to form PtdIns. However, the role each myotubularin family member plays in regulating endosomal PtdIns(3)P and thereby endocytic trafficking is not well established. Here, we identify the myotubularin family member MTMR4, which localizes to early endosomes and also to Rab11- and Sec15-positive recycling endosomes. In cells with MTMR4 knockdown, or following expression of the catalytically inactive MTMR4, MTMR4(C407A), the number of PtdIns(3)P-decorated endosomes significantly increased. MTMR4 overexpression delayed the exit of Tfn from early endosomes and its recycling to the plasma membrane. By contrast, expression of MTMR4(C407A), which acts as a dominant-negative construct, significantly accelerated Tfn recycling. However, in MTMR4 knockdown cells Tfn recycling was unchanged, suggesting that other MTMs might also contribute to recycling. MTMR4 regulated the subcellular distribution of Rab11 and, in cells with RNAi-mediated knockdown of MTMR4, Rab11 was directed away from the pericentriolar recycling compartment. The subcellular distribution of VAMP3, a v-SNARE protein that resides in recycling endosomes and endosome-derived transport vesicles, was also regulated by MTMR4. Therefore, MTMR4 localizes at the interface of early and recycling endosomes to regulate trafficking through this pathway.

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{beta}-Dystroglycan binds caveolin-1 in smooth muscle: a functional role in caveolae distribution and Ca2+ release.

Publication Date: 2010 Sep 15 PMID: 20736308
Authors: Sharma, P. - Ghavami, S. - Stelmack, G. L. - McNeill, K. D. - Mutawe, M. M. - Klonisch, T. - Unruh, H. - Halayko, A. J.
Journal: J Cell Sci

The dystrophin-glycoprotein complex (DGC) links the extracellular matrix and actin cytoskeleton. Caveolae form membrane arrays on smooth muscle cells; we investigated the mechanism for this organization. Caveolin-1 and beta-dystroglycan, the core transmembrane DGC subunit, colocalize in airway smooth muscle. Immunoprecipitation revealed the association of caveolin-1 with beta-dystroglycan. Disruption of actin filaments disordered caveolae arrays, reduced association of beta-dystroglycan and caveolin-1 to lipid rafts, and suppressed the sensitivity and responsiveness of methacholine-induced intracellular Ca(2+) release. We generated novel human airway smooth muscle cell lines expressing shRNA to stably silence beta-dystroglycan expression. In these myocytes, caveolae arrays were disorganized, caveolae structural proteins caveolin-1 and PTRF/cavin were displaced, the signaling proteins PLCbeta1 and G(alphaq), which are required for receptor-mediated Ca(2+) release, were absent from caveolae, and the sensitivity and responsiveness of methacholine-induced intracellular Ca(2+) release, was diminished. These data reveal an interaction between caveolin-1 and beta-dystroglycan and demonstrate that this association, in concert with anchorage to the actin cytoskeleton, underpins the spatial organization and functional role of caveolae in receptor-mediated Ca(2+) release, which is an essential initiator step in smooth muscle contraction.

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ERK5 is required for VEGF-mediated survival and tubular morphogenesis of primary human microvascular endothelial cells.

Publication Date: 2010 Sep 15 PMID: 20736307
Authors: Roberts, O. L. - Holmes, K. - Muller, J. - Cross, D. A. - Cross, M. J.
Journal: J Cell Sci

Extracellular signal-regulated kinase 5 (ERK5) is activated in response to environmental stress and growth factors. Gene ablation of Erk5 in mice is embryonically lethal as a result of disruption of cardiovascular development and vascular integrity. We investigated vascular endothelial growth factor (VEGF)-mediated ERK5 activation in primary human dermal microvascular endothelial cells (HDMECs) undergoing proliferation on a gelatin matrix, and tubular morphogenesis within a collagen gel matrix. VEGF induced sustained ERK5 activation on both matrices. However, manipulation of ERK5 activity by siRNA-mediated gene silencing disrupted tubular morphogenesis without impacting proliferation. Overexpression of constitutively active MEK5 and ERK5 stimulated tubular morphogenesis in the absence of VEGF. Analysis of intracellular signalling revealed that ERK5 regulated AKT phosphorylation. On a collagen gel, ERK5 regulated VEGF-mediated phosphorylation of the pro-apoptotic protein BAD and increased expression of the anti-apoptotic protein BCL2, resulting in decreased caspase-3 activity and apoptosis suppression. Our findings suggest that ERK5 is required for AKT phosphorylation and cell survival and is crucial for endothelial cell differentiation in response to VEGF.

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Formation of extra centrosomal structures is dependent on {beta}-catenin.

Publication Date: 2010 Sep 15 PMID: 20736306
Authors: Bahmanyar, S. - Guiney, E. L. - Hatch, E. M. - Nelson, W. J. - Barth, A. I.
Journal: J Cell Sci

beta-Catenin has important roles in cell-cell adhesion and in the regulation of gene transcription. Mutations that stabilize beta-catenin are common in cancer, but it remains unclear how these mutations contribute to cancer progression. beta-Catenin is also a centrosomal component involved in centrosome separation. Centrosomes nucleate interphase microtubules and the bipolar mitotic spindle in normal cells, but their organization and function in human cancers are abnormal. Here, we show that expression of stabilized mutant beta-catenin, which mimics mutations found in cancer, results in extra non-microtubule nucleating structures that contain a subset of centrosome proteins including gamma-tubulin and centrin, but not polo-like kinase 4 (Plk4), SAS-6 or pericentrin. A transcriptionally inactive form of beta-catenin also gives rise to abnormal structures of centrosome proteins. HCT116 human colon cancer cell lines, from which the mutant beta-catenin allele has been deleted, have reduced numbers of cells with abnormal centrosome structures and S-phase-arrested, amplified centrosomes. RNAi-mediated depletion of beta-catenin from centrosomes inhibits S-phase-arrested amplification of centrosomes. These results indicate that beta-catenin is required for centrosome amplification, and mutations in beta-catenin might contribute to the formation of abnormal centrosomes observed in cancers.

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SPICE - a previously uncharacterized protein required for centriole duplication and mitotic chromosome congression.

Publication Date: 2010 Sep 15 PMID: 20736305
Authors: Archinti, M. - Lacasa, C. - Teixido-Travesa, N. - Luders, J.
Journal: J Cell Sci

Proper assembly and function of a bipolar mitotic spindle is crucial for faithful bidirectional chromosome segregation during cell division. In animal cells, the two poles of the mitotic spindle are organized by centrosomes, microtubule-organizing structures composed of a pair of centrioles surrounded by the so-called pericentriolar material. Proteomic studies have revealed a large number of centrosome proteins, but many remain uncharacterized. Here, we characterize SPICE, a protein that localizes to spindle microtubules in mitosis and to centrioles throughout the cell cycle. RNAi-mediated depletion of SPICE in human cells impairs centriole duplication and causes severe mitotic defects. SPICE depletion compromises spindle architecture, spindle pole integrity and chromosome congression, even in cells in which centriole duplication has occurred. Our data suggest that SPICE is an important dual-function regulator required for centriole duplication and for proper bipolar spindle formation and chromosome congression in mitosis.

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Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry.

Publication Date: 2010 Sep 15 PMID: 20736304
Authors: Pozo-Guisado, E. - Campbell, D. G. - Deak, M. - Alvarez-Barrientos, A. - Morrice, N. A. - Alvarez, I. S. - Alessi, D. R. - Martin-Romero, F. J.
Journal: J Cell Sci

Store-operated calcium entry (SOCE) is an important Ca(2+) entry pathway that regulates many cell functions. Upon store depletion, STIM1, a transmembrane protein located in the endoplasmic reticulum (ER), aggregates and relocates close to the plasma membrane (PM) where it activates store-operated calcium channels (SOCs). Although STIM1 was early defined as a phosphoprotein, the contribution of the phosphorylation has been elusive. In the present work, STIM1 was found to be a target of extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in vitro, and we have defined the ERK1/2-phosphorylated sites on the STIM1 sequence. Using HEK293 cells stably transfected for the expression of tagged STIM1, we found that alanine substitution mutants of ERK1/2 target sites reduced SOCE significantly, suggesting that phosphorylation of these residues are required to fully accomplish SOCE. Indeed, the ERK1/2 inhibitors PD184352 and PD0325901 decreased SOCE in transfected cells. Conversely, 12-O-tetradecanoylphorbol-13-acetate, which activates ERK1/2, enhanced SOCE in cells expressing wild-type tagged STIM1, but did not potentiate Ca(2+) influx in cells expressing serine to alanine mutations in ERK1/2 target sites of STIM1. Alanine substitution mutations decreased Ca(2+) influx without disturbing the aggregation of STIM1 upon store depletion and without affecting the relocalization in ER-PM punctae. However, our results suggest that STIM1 phosphorylation at ERK1/2 target sites can modulate SOCE by altering STIM1 binding to SOCs, because a significant decrease in FRET efficiency was observed between alanine substitution mutants of STIM1-GFP and ORAI1-CFP.

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Leiomodin-2 is an antagonist of tropomodulin-1 at the pointed end of the thin filaments in cardiac muscle.

Publication Date: 2010 Sep 15 PMID: 20736303
Authors: Tsukada, T. - Pappas, C. T. - Moroz, N. - Antin, P. B. - Kostyukova, A. S. - Gregorio, C. C.
Journal: J Cell Sci

Regulation of actin filament assembly is essential for efficient contractile activity in striated muscle. Leiomodin is an actin-binding protein and homolog of the pointed-end capping protein, tropomodulin. These proteins are structurally similar, sharing a common domain organization that includes two actin-binding sites. Leiomodin also contains a unique C-terminal extension that has a third actin-binding WH2 domain. Recently, the striated-muscle-specific isoform of leiomodin (Lmod2) was reported to be an actin nucleator in cardiomyocytes. Here, we have identified a function of Lmod2 in the regulation of thin filament lengths. We show that Lmod2 localizes to the pointed ends of thin filaments, where it competes for binding with tropomodulin-1 (Tmod1). Overexpression of Lmod2 results in loss of Tmod1 assembly and elongation of the thin filaments from their pointed ends. The Lmod2 WH2 domain is required for lengthening because its removal results in a molecule that caps the pointed ends similarly to Tmod1. Furthermore, Lmod2 transcripts are first detected in the heart after it has begun to beat, suggesting that the primary function of Lmod2 is to maintain thin filament lengths in the mature heart. Thus, Lmod2 antagonizes the function of Tmod1, and together, these molecules might fine-tune thin filament lengths.

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Regulation of mixed-lineage kinase activation in JNK-dependent morphogenesis.

Publication Date: 2010 Sep 15 PMID: 20736302
Authors: Garlena, R. A. - Gonda, R. L. - Green, A. B. - Pileggi, R. M. - Stronach, B.
Journal: J Cell Sci

Normal cells respond appropriately to various signals, while sustaining proper developmental programs and tissue homeostasis. Inappropriate signal reception, response or attenuation, can upset the normal balance of signaling within cells, leading to dysfunction or tissue malformation. To understand the molecular mechanisms that regulate protein-kinase-based signaling in the context of tissue morphogenesis, we analyzed the domain requirements of Drosophila Slpr, a mixed-lineage kinase (MLK), for Jun N-terminal kinase (JNK) signaling. The N-terminal half of Slpr is involved in regulated signaling whereas the C-terminal half promotes cortical protein localization. The SH3 domain negatively regulates Slpr activity consistent with autoinhibition via a conserved proline motif. Also, like many kinases, conserved residues in the activation segment of the catalytic domain regulate Slpr. Threonine 295, in particular, is essential for function. Slpr activation requires dual input from the MAP4K Misshapen (Msn), through its C-terminal regulatory domain, and the GTPase Rac, which both bind to the LZ-CRIB region of Slpr in vitro. Although Rac is sufficient to activate JNK signaling, our results indicate that there are Slpr-independent functions for Rac in dorsal closure. Finally, expression of various Slpr constructs alone or with upstream activators reveals a wide-ranging response at the cell and tissue level.

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Chemokine expression and control of muscle cell migration during myogenesis.

Publication Date: 2010 Sep 15 PMID: 20736301
Authors: Griffin, C. A. - Apponi, L. H. - Long, K. K. - Pavlath, G. K.
Journal: J Cell Sci

Adult regenerative myogenesis is vital for restoring normal tissue structure after muscle injury. Muscle regeneration is dependent on progenitor satellite cells, which proliferate in response to injury, and their progeny differentiate and undergo cell-cell fusion to form regenerating myofibers. Myogenic progenitor cells must be precisely regulated and positioned for proper cell fusion to occur. Chemokines are secreted proteins that share both leukocyte chemoattractant and cytokine-like behavior and affect the physiology of a number of cell types. We investigated the steady-state mRNA levels of 84 chemokines, chemokine receptors and signaling molecules, to obtain a comprehensive view of chemokine expression by muscle cells during myogenesis in vitro. A large number of chemokines and chemokine receptors were expressed by primary mouse muscle cells, especially during times of extensive cell-cell fusion. Furthermore, muscle cells exhibited different migratory behavior throughout myogenesis in vitro. One receptor-ligand pair, CXCR4-SDF-1alpha (CXCL12), regulated migration of both proliferating and terminally differentiated muscle cells, and was necessary for proper fusion of muscle cells. Given the large number of chemokines and chemokine receptors directly expressed by muscle cells, these proteins might have a greater role in myogenesis than previously appreciated.

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The fibroblast-derived paracrine factor neuregulin-1 has a novel role in regulating the constitutive color and melanocyte function in human skin.

Publication Date: 2010 Sep 15 PMID: 20736300
Authors: Choi, W. - Wolber, R. - Gerwat, W. - Mann, T. - Batzer, J. - Smuda, C. - Liu, H. - Kolbe, L. - Hearing, V. J.
Journal: J Cell Sci

Interactions between melanocytes and neighboring cells in the skin are important in regulating skin color in humans. We recently demonstrated that the less pigmented and thicker skin on the palms and soles is regulated by underlying fibroblasts in those areas, specifically via a secreted factor (DKK1) that modulates Wnt signaling. In this study, we tested the hypothesis that dermal fibroblasts regulate the constitutive skin color of individuals ranging from very light to very dark. We used microarray analysis to compare gene expression patterns in fibroblasts derived from lighter skin types compared to darker skin types, with a focus on secreted proteins. We identified a number of genes that differ dramatically in expression and, among the expressed proteins, neuregulin-1, which is secreted by fibroblasts derived from dark skin, effectively increases the pigmentation of melanocytes in tissue culture and in an artificial skin model and regulates their growth, suggesting that it is one of the major factors determining human skin color.

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Protein mobilities and P-selectin storage in Weibel-Palade bodies.

Publication Date: 2010 Sep 1 PMID: 20720153
Authors: Kiskin, N. I. - Hellen, N. - Babich, V. - Hewlett, L. - Knipe, L. - Hannah, M. J. - Carter, T.
Journal: J Cell Sci

Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel-Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P-selectin became immobilised, whereas small proteins (ssEGFP, eotaxin-3) became less mobile. WPB maturation led to further decreases in mobility of small proteins and CD63. Acute alkalinisation of mature WPBs selectively increased the mobilities of small soluble proteins without affecting larger molecules and the membrane proteins. Disruption of the Proregion-VWF paracrystalline core by prolonged incubation with NH(4)Cl rendered P-selectin mobile while VWF remained immobile. FRAP of P-selectin mutants revealed that immobilisation most probably involves steric entrapment of the P-selectin extracellular domain by the Proregion-VWF paracrystal. Significantly, immobilisation contributed to the enrichment of P-selectin in WPBs; a mutation of P-selectin preventing immobilisation led to a failure of enrichment. Together these data shed new light on the transitions that occur for soluble and membrane proteins following their entry and storage into post-Golgi-regulated secretory organelles.

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Cytoskeletal dysfunction dominates in DAP12-deficient osteoclasts.

Publication Date: 2010 Sep 1 PMID: 20720152
Authors: Zou, W. - Zhu, T. - Craft, C. S. - Broekelmann, T. J. - Mecham, R. P. - Teitelbaum, S. L.
Journal: J Cell Sci

Despite evidence that DAP12 regulates osteoclasts, mice lacking the ITAM-bearing protein exhibit only mild osteopetrosis. Alternatively, Dap12(-/-) mice, also lacking FcRgamma, are severely osteopetrotic, suggesting that FcRgamma compensates for DAP12 deficiency in the bone-resorbing polykaryons. Controversy exists, however, as to whether these co-stimulatory molecules regulate differentiation of osteoclasts or the capacity of the mature cell to degrade bone. We find that Dap12(-/-) osteoclasts differentiate normally when generated on osteoblasts but have a dysfunctional cytoskeleton, impairing their ability to transmigrate through the osteoblast layer and resorb bone. To determine whether the FcRgamma co-receptor, OSCAR mediates osteoclast function in the absence of DAP12, we overexpressed OSCAR fused to FLAG (OSCAR-FLAG), in Dap12(-/-) osteoclasts. OSCAR-FLAG partially rescues the abnormal cytoskeleton of Dap12(-/-) osteoclasts grown on bone, but not those grown on osteoblasts. Thus, cytoskeletal dysfunction, and not arrested differentiation, is the dominant consequence of DAP12 deficiency in osteoclasts. The failure of osteoblasts to normalize Dap12(-/-) osteoclasts indicates that functionally relevant quantities of OSCAR ligand do not reside in bone-forming cells.

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Control of Notch-ligand endocytosis by ligand-receptor interaction.

Publication Date: 2010 Sep 1 PMID: 20720151
Authors: Hansson, E. M. - Lanner, F. - Das, D. - Mutvei, A. - Marklund, U. - Ericson, J. - Farnebo, F. - Stumm, G. - Stenmark, H. - Andersson, E. R. - Lendahl, U.
Journal: J Cell Sci

In Notch signaling, cell-bound ligands activate Notch receptors on juxtaposed cells, but the relationship between ligand endocytosis, ubiquitylation and ligand-receptor interaction remains poorly understood. To study the specific role of ligand-receptor interaction, we identified a missense mutant of the Notch ligand Jagged1 (Nodder, Ndr) that failed to interact with Notch receptors, but retained a cellular distribution that was similar to wild-type Jagged1 (Jagged1(WT)) in the absence of active Notch signaling. Both Jagged1(WT) and Jagged1(Ndr) interacted with the E3 ubiquitin ligase Mind bomb, but only Jagged1(WT) showed enhanced ubiquitylation after co-culture with cells expressing Notch receptor. Cells expressing Jagged1(WT), but not Jagged1(Ndr), trans-endocytosed the Notch extracellular domain (NECD) into the ligand-expressing cell, and NECD colocalized with Jagged1(WT) in early endosomes, multivesicular bodies and lysosomes, suggesting that NECD is routed through the endocytic degradation pathway. When coexpressed in the same cell, Jagged1(Ndr) did not exert a dominant-negative effect over Jagged1(WT) in terms of receptor activation. Finally, in Jag1(Ndr/Ndr) mice, the ligand was largely accumulated at the cell surface, indicating that engagement of the Notch receptor is important for ligand internalization in vivo. In conclusion, the interaction-dead Jagged1(Ndr) ligand provides new insights into the specific role of receptor-ligand interaction in the intracellular trafficking of Notch ligands.

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Cdc14: a highly conserved family of phosphatases with non-conserved functions?

Publication Date: 2010 Sep 1 PMID: 20720150
Authors: Mocciaro, A. - Schiebel, E.
Journal: J Cell Sci

CDC14 was originally identified by L. Hartwell in his famous screen for genes that regulate the budding yeast cell cycle. Subsequent work showed that Cdc14 belongs to a family of highly conserved dual-specificity phosphatases that are present in a wide range of organisms from yeast to human. Human CDC14B is even able to fulfill the essential functions of budding yeast Cdc14. In budding yeast, Cdc14 counteracts the activity of cyclin dependent kinase (Cdk1) at the end of mitosis and thus has important roles in the regulation of anaphase, mitotic exit and cytokinesis. On the basis of the functional conservation of other cell-cycle genes it seemed obvious to assume that Cdc14 phosphatases also have roles in late mitosis in mammalian cells and regulate similar targets to those found in yeast. However, analysis of the human Cdc14 proteins (CDC14A, CDC14B and CDC14C) by overexpression or by depletion using small interfering RNA (siRNA) has suggested functions that are quite different from those of ScCdc14. Recent studies in avian and human somatic cell lines in which the gene encoding either Cdc14A or Cdc14B had been deleted, have shown - surprisingly - that neither of the two phosphatases on its own is essential for viability, cell-cycle progression and checkpoint control. In this Commentary, we critically review the available data on the functions of yeast and vertebrate Cdc14 phosphatases, and discuss whether they indeed share common functions as generally assumed.

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Lumican is required for neutrophil extravasation following corneal injury and wound healing.

Publication Date: 2010 Sep 1 PMID: 20699360
Authors: Hayashi, Y. - Call, M. K. - Chikama, T. - Liu, H. - Carlson, E. C. - Sun, Y. - Pearlman, E. - Funderburgh, J. L. - Babcock, G. - Liu, C. Y. - Ohashi, Y. - Kao, W. W.
Journal: J Cell Sci

An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes, including cell proliferation and migration. In the present study, we addressed the role of lumican in the process of extravasation of polymorphonuclear leukocytes (PMNs) during the early inflammatory phase present in the healing of the corneal epithelium following debridement. We used Lum(-/-) mice and a novel transgenic mouse, Lum(-/-),Kera-Lum, which expresses lumican only in the corneal stroma, to assess the role of lumican in PMN extravasation into injured corneas. Our results showed that PMNs did not readily invade injured corneas of Lum(-/-) mice and this defect was rescued by the expression of lumican in the corneas of Lum(-/-),Kera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity in casein-induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen fibril architecture, lumican acts to aid neutrophil recruitment and invasion following corneal damage and inflammation.

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Casein kinase 1 regulates human hypoxia-inducible factor HIF-1.

Publication Date: 2010 Sep 1 PMID: 20699359
Authors: Kalousi, A. - Mylonis, I. - Politou, A. S. - Chachami, G. - Paraskeva, E. - Simos, G.
Journal: J Cell Sci

Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular response to hypoxia and a promising target of anticancer therapy, is essential for adaptation to low oxygen conditions, embryogenesis and tumor progression. HIF-1 is a heterodimer of HIF-1alpha, expression of which is controlled by oxygen levels as well as by various oxygen-independent mechanisms, and HIF-1beta (or ARNT), which is constitutively expressed. In this work, we investigate the phosphorylation of the N-terminal heterodimerization (PAS) domain of HIF-1alpha and identify Ser247 as a major site of in vitro modification by casein kinase 1delta (CK1delta). Mutation of this site to alanine, surprisingly, enhanced the transcriptional activity of HIF-1alpha, a result phenocopied by inhibition or small interfering RNA (siRNA)-mediated silencing of CK1delta under hypoxic conditions. Conversely, overexpression of CK1delta or phosphomimetic mutation of Ser247 to aspartate inhibited HIF-1alpha activity without affecting its stability or nuclear accumulation. Immunoprecipitation and in vitro binding experiments suggest that CK1-dependent phosphorylation of HIF-1alpha at Ser247 impairs its association with ARNT, a notion also supported by modeling the structure of the complex between HIF-1alpha and ARNT PAS-B domains. We suggest that modification of HIF-1alpha by CK1 represents a novel mechanism that controls the activity of HIF-1 during hypoxia by regulating the interaction between its two subunits.

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14-3-3 sigma associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1.

Publication Date: 2010 Sep 1 PMID: 20699358
Authors: Ghaffari, A. - Li, Y. - Kilani, R. T. - Ghahary, A.
Journal: J Cell Sci

Matrix metalloproteinases (MMPs) are implicated in the degradation of the extracellular matrix during development and tissue repair, as well as in pathological conditions such as tumor invasion and fibrosis. MMP expression by stromal cells is partly regulated by signals from the neighboring epithelial cells. Keratinocyte-releasable 14-3-3sigma, or stratifin, acts as a potent MMP-1-stimulatory factor in fibroblasts. However, its mechanism of transmembrane signaling remains unknown. Ectodomain biotin labeling, serial affinity purification and mass spectroscopy analysis revealed that the stratifin associates with aminopeptidase N (APN), or CD13, at the cell surface. The transient knockdown of APN in fibroblasts eliminated the stratifin-mediated p38 MAP kinase activation and MMP-1 expression, implicating APN in a receptor-mediated transmembrane signaling event. Stratifin deletion studies implicated its C-terminus as a potential APN-binding site. Furthermore, the dephosphorylation of APN ectodomains reduced its binding affinity to the stratifin. The presence of a phosphorylated serine or threonine residue in APN has been implicated. Together, these findings provide evidence that APN is a novel cell surface receptor for stratifin and a potential target in the regulation of MMP-1 expression in epithelial-stromal cell communication.

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Assembly of fibrillin microfibrils governs extracellular deposition of latent TGF beta.

Publication Date: 2010 Sep 1 PMID: 20699357
Authors: Massam-Wu, T. - Chiu, M. - Choudhury, R. - Chaudhry, S. S. - Baldwin, A. K. - McGovern, A. - Baldock, C. - Shuttleworth, C. A. - Kielty, C. M.
Journal: J Cell Sci

Control of the bioavailability of the growth factor TGFbeta is essential for tissue formation and homeostasis, yet precisely how latent TGFbeta is incorporated into the extracellular matrix is unknown. Here, we show that deposition of a large latent TGFbeta complex (LLC), which contains latent TGFbeta-binding protein 1 (LTBP-1), is directly dependent on the pericellular assembly of fibrillin microfibrils, which interact with fibronectin during higher-order fibrillogenesis. LTBP-1 formed pericellular arrays that colocalized with microfibrils, whereas fibrillin knockdown inhibited fibrillar LTBP-1 and/or LLC deposition. Blocking alpha5beta1 integrin or supplementing cultures with heparin, which both inhibited microfibril assembly, disrupted LTBP-1 deposition and enhanced Smad2 phosphorylation. Full-length LTBP-1 bound only weakly to N-terminal pro-fibrillin-1, but this association was strongly enhanced by heparin. The microfibril-associated glycoprotein MAGP-1 (MFAP-2) inhibited LTBP-1 binding to fibrillin-1 and stimulated Smad2 phosphorylation. By contrast, fibulin-4, which interacted strongly with full-length LTBP-1, did not induce Smad2 phosphorylation. Thus, LTBP-1 and/or LLC deposition is dependent on pericellular microfibril assembly and is governed by complex interactions between LTBP-1, heparan sulfate, fibrillin-1 and microfibril-associated molecules. In this way, microfibrils control TGFbeta bioavailability.

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Strategies of actin reorganisation in plant cells.

Publication Date: 2010 Sep 1 PMID: 20699356
Authors: Smertenko, A. P. - Deeks, M. J. - Hussey, P. J.
Journal: J Cell Sci

Spatial-temporal flexibility of the actin filament network (F-actin) is essential for all basic cellular functions and is governed by a stochastic dynamic model. In this model, actin filaments that randomly polymerise from a pool of free actin are bundled with other filaments and severed by ADF/cofilin. The fate of the severed fragments is not known. It has been proposed that the fragments are disassembled and the monomeric actin recycled for the polymerisation of new filaments. Here, we have generated tobacco cell lines and Arabidopsis plants expressing the actin marker Lifeact to address the mechanisms of F-actin reorganisation in vivo. We found that F-actin is more dynamic in isotropically expanding cells and that the density of the network changes with a periodicity of 70 seconds. The depolymerisation rate, but not the polymerisation rate, of F-actin increases when microtubules are destabilised. New filaments can be assembled from shorter free cytoplasmic fragments, from the products of F-actin severing and by polymerisation from the ends of extant filaments. Thus, remodelling of F-actin might not require bulk depolymerisation of the entire network, but could occur via severing and end-joining of existing polymers.

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CHMP2B mutants linked to frontotemporal dementia impair maturation of dendritic spines.

Publication Date: 2010 Sep 1 PMID: 20699355
Authors: Belly, A. - Bodon, G. - Blot, B. - Bouron, A. - Sadoul, R. - Goldberg, Y.
Journal: J Cell Sci

The highly conserved ESCRT-III complex is responsible for deformation and cleavage of membranes during endosomal trafficking and other cellular activities. In humans, dominant mutations in the ESCRT-III subunit CHMP2B cause frontotemporal dementia (FTD). The decade-long process leading to this cortical degeneration is not well understood. One possibility is that, akin to other neurodegenerative diseases, the pathogenic protein affects the integrity of dendritic spines and synapses before any neuronal death. Using confocal microscopy and 3D reconstruction, we examined whether expressing the FTD-linked mutants CHMP2B(intron5) and CHMP2B(Delta10) in cultured hippocampal neurons modified the number or structure of spines. Both mutants induced a significant decrease in the proportion of large spines with mushroom morphology, without overt degeneration. Furthermore, CHMP2B(Delta10) induced a drop in frequency and amplitude of spontaneous excitatory postsynaptic currents, suggesting that the more potent synapses were lost. These effects seemed unrelated to changes in autophagy. Depletion of endogenous CHMP2B by RNAi resulted in morphological changes similar to those induced by mutant CHMP2B, consistent with dominant-negative activity of pathogenic mutants. Thus, CHMP2B is required for spine growth. Taken together, these results demonstrate that a mutant ESCRT-III subunit linked to a human neurodegenerative disease can disrupt the normal pattern of spine development.

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TIMP3: a physiological regulator of adult myogenesis.

Publication Date: 2010 Sep 1 PMID: 20682640
Authors: Liu, H. - Chen, S. E. - Jin, B. - Carson, J. A. - Niu, A. - Durham, W. - Lai, J. Y. - Li, Y. P.
Journal: J Cell Sci

Myogenic differentiation in adult muscle is normally suppressed and can be activated by myogenic cues in a subset of activated satellite cells. The switch mechanism that turns myogenesis on and off is not defined. In the present study, we demonstrate that tissue inhibitor of metalloproteinase 3 (TIMP3), the endogenous inhibitor of TNFalpha-converting enzyme (TACE), acts as an on-off switch for myogenic differentiation by regulating autocrine TNFalpha release. We observed that constitutively expressed TIMP3 is transiently downregulated in the satellite cells of regenerating mouse hindlimb muscles and differentiating C2C12 myoblasts. In C2C12 myoblasts, perturbing TIMP3 downregulation by overexpressing TIMP3 blocks TNFalpha release, p38 MAPK activation, myogenic gene expression and myotube formation. TNFalpha supplementation at a physiological concentration rescues myoblast differentiation. Similarly, in the regenerating soleus, overexpression of TIMP3 impairs release of TNFalpha and myogenic gene expression, and delays the formation of new fibers. In addition, downregulation of TIMP3 is mediated by the myogenesis-promoting microRNA miR-206. Thus, TIMP3 is a physiological regulator of myogenic differentiation.

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Mobility of G proteins is heterogeneous and polarized during chemotaxis.

Publication Date: 2010 Sep 1 PMID: 20682639
Authors: van Hemert, F. - Lazova, M. D. - Snaar-Jagaska, B. E. - Schmidt, T.
Journal: J Cell Sci

The interaction of G-protein-coupled receptors with G proteins is a key event in transmembrane signal transduction that leads to vital decision-making by the cell. Here, we applied single-molecule epifluorescence microscopy to study the mobility of both the Gbetagamma and the Galpha2 subunits of the G protein heterotrimer in comparison with the cAMP receptor responsible for chemotactic signaling in Dictyostelium discoideum. Our experimental results suggest that approximately 30% of the G protein heterotrimers exist in receptor-precoupled complexes. Upon stimulation in a chemotactic gradient, this complex dissociates, subsequently leading to a linear diffusion and collision amplification of the external signal. We further found that Gbetagamma was partially immobilized and confined in an agonist-, F-actin- and Galpha2-dependent fashion. This led to the hypothesis that functional nanometric domains exist in the plasma membrane, which locally restrict the activation signal, and in turn, lead to faithful and efficient chemotactic signaling.

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Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response.

Publication Date: 2010 Sep 1 PMID: 20682638
Authors: Malzer, E. - Daly, M. L. - Moloney, A. - Sendall, T. J. - Thomas, S. E. - Ryder, E. - Ryoo, H. D. - Crowther, D. C. - Lomas, D. A. - Marciniak, S. J.
Journal: J Cell Sci

The integrated stress response (ISR) protects cells from numerous forms of stress and is involved in the growth of solid tumours; however, it is unclear how the ISR acts on cellular proliferation. We have developed a model of ISR signalling with which to study its effects on tissue growth. Overexpression of the ISR kinase PERK resulted in a striking atrophic eye phenotype in Drosophila melanogaster that could be rescued by co-expressing the eIF2alpha phosphatase GADD34. A genetic screen of 3000 transposon insertions identified grapes, the gene that encodes the Drosophila orthologue of checkpoint kinase 1 (CHK1). Knockdown of grapes by RNAi rescued eye development despite ongoing PERK activation. In mammalian cells, CHK1 was activated by agents that induce ER stress, which resulted in a G2 cell cycle delay. PERK was both necessary and sufficient for CHK1 activation. These findings indicate that non-genotoxic misfolded protein stress accesses DNA-damage-induced cell cycle checkpoints to couple the ISR to cell cycle arrest.

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Basal body movements orchestrate membrane organelle division and cell morphogenesis in Trypanosoma brucei.

Publication Date: 2010 Sep 1 PMID: 20682637
Authors: Lacomble, S. - Vaughan, S. - Gadelha, C. - Morphew, M. K. - Shaw, M. K. - McIntosh, J. R. - Gull, K.
Journal: J Cell Sci

The defined shape and single-copy organelles of Trypanosoma brucei mean that it provides an excellent model in which to study how duplication and segregation of organelles is interfaced with morphogenesis of overall cell shape and form. The centriole or basal body of eukaryotic cells is often seen to be at the centre of such processes. We have used a combination of electron microscopy and electron tomography techniques to provide a detailed three-dimensional view of duplication of the basal body in trypanosomes. We show that the basal body duplication and maturation cycle exerts an influence on the intimately associated flagellar pocket membrane system that is the portal for secretion and uptake from this cell. At the start of the cell cycle, a probasal body is positioned anterior to the basal body of the existing flagellum. At the G1-S transition, the probasal body matures, elongates and invades the pre-existing flagellar pocket to form the new flagellar axoneme. The new basal body undergoes a spectacular anti-clockwise rotation around the old flagellum, while its short new axoneme is associated with the pre-existing flagellar pocket. This rotation and subsequent posterior movements results in division of the flagellar pocket and ultimately sets parameters for subsequent daughter cell morphogenesis.

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Elevated expression of protein kinase C delta induces cell scattering upon serum deprivation.

Publication Date: 2010 Sep 1 PMID: 20682636
Authors: Chen, C. L. - Chan, P. C. - Wang, S. H. - Pan, Y. R. - Chen, H. C.
Journal: J Cell Sci

Tumor metastasis might be evoked in response to microenvironmental stress, such as a shortage of oxygen. Although the cellular response to hypoxia has been well established, we know little about how tumors adapt themselves to deprivation of growth factor. Protein kinase Cdelta (PKCdelta), a stress-sensitive protein kinase, has been implicated in tumor progression. In this study, we demonstrate that elevated expression of PKCdelta in Madin-Darby canine kidney cells induces a scatter response upon serum starvation, a condition that mimics growth-factor deprivation. Serum starvation stimulates the catalytic activity and Y311 phosphorylation of PKCdelta through reactive oxygen species (ROS) and the Src family kinases. Mutation of PKCdelta at Y311 and Y322, both of which are phosphorylation sites for Src, impairs its activation and ability to promote cell scattering upon serum deprivation. Once activated by ROS, PKCdelta itself activates ROS production at least partially through NADPH oxidase. In addition, the c-Jun N-terminal kinase is identified as a crucial downstream mediator of ROS and PKCdelta for induction of cell scattering upon serum deprivation. We demonstrate that the C1B domain of PKCdelta is essential not only for its localization at the Golgi complex, but also for its activation and ability to induce cell scattering upon serum deprivation. Finally, depletion of PKCdelta in human bladder carcinoma T24 cells restores their cell-cell contacts, which thereby reverses a scattered growth pattern to an epithelial-like growth pattern. Collectively, our results suggest that elevated expression of PKCdelta might facilitate the scattering of cells in order to escape stress induced by growth-factor deprivation.

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Decoupling diffusional from dimensional control of signaling in 3D culture reveals a role for myosin in tubulogenesis.

Publication Date: 2010 Sep 1 PMID: 20682635
Authors: Raghavan, S. - Shen, C. J. - Desai, R. A. - Sniadecki, N. J. - Nelson, C. M. - Chen, C. S.
Journal: J Cell Sci

We present a novel microfabricated platform to culture cells within arrays of micrometer-scale three-dimensional (3D) extracellular matrix scaffolds (microgels). These microscale cultures eliminate diffusion barriers that are intrinsic to traditional 3D culture systems (macrogels) and enable uniform cytokine stimulation of the entire culture population, as well as allow immunolabeling, imaging and population-based biochemical assays across the relatively coplanar microgels. Examining early signaling associated with hepatocyte growth factor (HGF)-mediated scattering and tubulogenesis of MDCK cells revealed that 3D culture modulates cellular responses both through dimensionality and altered stimulation rates. Comparing responses in 2D culture, microgels and macrogels demonstrated that HGF-induced ERK signaling was driven by the dynamics of stimulation and not by whether cells were in a 2D or 3D environment, and that this ERK signaling was equally important for HGF-induced cell scattering on 2D substrates and tubulogenesis in 3D. By contrast, we discovered a specific HGF-induced increase in myosin expression leading to sustained downregulation of myosin activity that occurred only within 3D contexts and was required for 3D tubulogenesis but not 2D scattering. Interestingly, although absent in cells on collagen-coated plates, downregulation of myosin activity also occurred for cells on collagen gels, but was transient and mediated by a combination of myosin dephosphorylation and enhanced myosin expression. Furthermore, upregulating myosin activity via siRNA targeted to a myosin phosphatase did not attenuate scattering in 2D but did inhibit tubulogenesis in 3D. Together, these results demonstrate that cellular responses to soluble cues in 3D culture are regulated by both rates of stimulation and by matrix dimensionality, and highlight the importance of decoupling these effects to identify early signals relevant to cellular function in 3D environments.

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E-cadherin regulates MAL-SRF-mediated transcription in epithelial cells.

Publication Date: 2010 Aug 15 PMID: 20663922
Authors: Busche, S. - Kremmer, E. - Posern, G.
Journal: J Cell Sci

Epithelial junctions are dynamically and functionally linked to the actin cytoskeleton, and their disassembly is a key event during physiological and pathological processes. We recently showed that epithelial disintegration facilitates transcriptional activation via Rac, G-actin, MAL (also known as MRTF) and serum response factor (SRF). Here, we investigate which specific component of the epithelial junction is essential for this MAL-SRF-mediated transcription. The Ca(2+)-dependent dissociation of polarised epithelial cells depleted of ZO proteins - which form adherens junctions (AJs) but completely lack tight junctions (TJs) - fully activated SRF. By contrast, AGS gastric adenocarcinoma epithelial cells, which form TJs but are deficient in E-cadherin, and therefore also in AJs, failed to activate SRF. The introduction of wild-type E-cadherin in AGS cells restored AJ formation and MAL-SRF inducibility. To gain further insight into the membrane-proximal signalling, AGS cells were stably transfected with E-cadherin-alpha-catenin fusions. Despite restored formation of cell-cell contacts containing the nectin-afadin complex and p120-catenin, these cells did not activate SRF upon junction dissociation, suggesting that signal transmission depends on the C-terminal tail of E-cadherin. We conclude that the dissociation of intercellular E-cadherin interactions from AJs, and signals originating from the C-terminal region covering the beta-catenin-binding site of E-cadherin, are essential for transcriptional activation via Rac, MAL and SRF, whereas TJs are not involved.

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LARP-1 promotes oogenesis by repressing fem-3 in the C. elegans germline.

Publication Date: 2010 Aug 15 PMID: 20663921
Authors: Zanin, E. - Pacquelet, A. - Scheckel, C. - Ciosk, R. - Gotta, M.
Journal: J Cell Sci

LA-related protein 1 (LARP-1) belongs to an RNA-binding protein family containing a LA motif. Here, we identify LARP-1 as a regulator of sex determination. In C. elegans hermaphrodites, a complex regulatory network regulates the switch from sperm to oocyte production. We find that simultaneous depletion of larp-1 and the Nanos homologue nos-3 results in germline masculinization. This phenotype is accompanied by a strong reduction of the levels of TRA-1, a GLI-family transcription factor that promotes oogenesis. TRA-1 levels are regulated by CBC(FEM-1), a ubiquitin ligase consisting of the FEM proteins, FEM-1, FEM-2 and FEM-3 and the cullin CUL-2. We show that both the masculinization phenotype and the reduction of TRA-1 levels observed in nos-3;larp-1 mutants require fem-3 activity, suggesting that nos-3 and larp-1 regulate the sperm-oocyte switch by inhibiting the fem genes. Consistently, fem-3 mRNA levels are increased in larp-1 mutants. By contrast, levels of fem-3 mRNA are not affected in nos-3 mutants. Therefore, our data indicate that LARP-1 and NOS-3 promote oogenesis by regulating fem-3 expression through distinct mechanisms.

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Identification and characterization of GRIM-1, a cell-death-associated gene product.

Publication Date: 2010 Aug 15 PMID: 20663920
Authors: Hofmann, E. R. - Nallar, S. C. - Lin, L. - D'Cunha, J. - Lindner, D. J. - Weihua, X. - Kalvakolanu, D. V.
Journal: J Cell Sci

Using a genome-wide technical knockout, we isolated a newly identified set of GRIM (genes associated with retinoid-interferon-induced mortality) genes; GRIM genes mediate IFN- and retinoic-acid (RA)-induced cell death. Here, we describe the isolation and characterization of one such gene, GRIM-1. Three proteins, with identical C-termini, were produced from the GRIM-1 open reading frame when this gene was transcribed and translated in vitro. These protein isoforms, designated GRIM-1alpha, GRIM-1beta and GRIM-1gamma, differentially suppressed growth via apoptosis in various cell lines. We also show that a caspase-dependent mechanism generates the proapoptotic GRIM-1 isoforms. Lastly, GRIM-1 isoforms differentially blocked maturation of 18S ribosomal RNA, consistent with their respective growth-suppressive ability. Together, these studies identified a novel protein involved in growth suppression and cell death.

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Activation of pancreatic-duct-derived progenitor cells during pancreas regeneration in adult rats.

Publication Date: 2010 Aug 15 PMID: 20663919
Authors: Li, W. C. - Rukstalis, J. M. - Nishimura, W. - Tchipashvili, V. - Habener, J. F. - Sharma, A. - Bonner-Weir, S.
Journal: J Cell Sci

The adult pancreas has considerable capacity to regenerate in response to injury. We hypothesized that after partial pancreatectomy (Px) in adult rats, pancreatic-duct cells serve as a source of regeneration by undergoing a reproducible dedifferentiation and redifferentiation. We support this hypothesis by the detection of an early loss of the ductal differentiation marker Hnf6 in the mature ducts, followed by the transient appearance of areas composed of proliferating ductules, called foci of regeneration, which subsequently form new pancreatic lobes. In young foci, ductules express markers of the embryonic pancreatic epithelium - Pdx1, Tcf2 and Sox9 - suggesting that these cells act as progenitors of the regenerating pancreas. The endocrine-lineage-specific transcription factor Neurogenin3, which is found in the developing embryonic pancreas, was transiently detected in the foci. Islets in foci initially resemble embryonic islets in their lack of MafA expression and lower percentage of beta-cells, but with increasing maturation have increasing numbers of MafA(+) insulin(+) cells. Taken together, we provide a mechanism by which adult pancreatic duct cells recapitulate aspects of embryonic pancreas differentiation in response to injury, and contribute to regeneration of the pancreas. This mechanism of regeneration relies mainly on the plasticity of the differentiated cells within the pancreas.

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Physiological levels of TNFalpha stimulation induce stochastic dynamics of NF-kappaB responses in single living cells.

Publication Date: 2010 Aug 15 PMID: 20663918
Authors: Turner, D. A. - Paszek, P. - Woodcock, D. J. - Nelson, D. E. - Horton, C. A. - Wang, Y. - Spiller, D. G. - Rand, D. A. - White, M. R. - Harper, C. V.
Journal: J Cell Sci

Nuclear factor kappa B (NF-kappaB) signalling is activated by cellular stress and inflammation and regulates cytokine expression. We applied single-cell imaging to investigate dynamic responses to different doses of tumour necrosis factor alpha (TNFalpha). Lower doses activated fewer cells and those responding showed an increasingly variable delay in the initial NF-kappaB nuclear translocation and associated IkappaBalpha degradation. Robust 100 minute nuclear:cytoplasmic NF-kappaB oscillations were observed over a wide range of TNFalpha concentrations. The result is supported by computational analyses, which identified a limit cycle in the system with a stable 100 minute period over a range of stimuli, and indicated no co-operativity in the pathway activation. These results suggest that a stochastic threshold controls functional all-or-nothing responses in individual cells. Deterministic and stochastic models simulated the experimentally observed activation threshold and gave rise to new predictions about the structure of the system and open the way for better mechanistic understanding of physiological TNFalpha activation of inflammatory responses in cells and tissues.

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E1--E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium.

Publication Date: 2010 Aug 15 PMID: 20663917
Authors: McIntosh, P. B. - Laskey, P. - Sullivan, K. - Davy, C. - Wang, Q. - Jackson, D. J. - Griffin, H. M. - Doorbar, J.
Journal: J Cell Sci

The keratin IF network of epidermal keratinocytes provides a protective barrier against mechanical insult, it is also a major player in absorbing stress in these cells. The human papilloma virus (HPV) type 16 E1--E4 protein accumulates in the upper layers of HPV16-infected epithelium and is known to associate with and reorganise the keratin IF network in cells in culture. Here, we show that this function is conserved amongst a number of HPV alpha-group E1--E4 proteins and that the differentiation-dependent keratins are also targeted. Using time-lapse microscopy, HPV16 E1--E4 was found to effect a dramatic cessation of keratin IF network dynamics by associating with both soluble and insoluble keratin. Network disruption was accompanied by keratin hyperphosphorylation at several sites, including K8 S73, which is typically phosphorylated in response to stress stimuli. Keratin immunoprecipitated from E1--E4-expressing cells was also found to be ubiquitylated, indicating that it is targeted for proteasomal degradation. Interestingly, the accumulation of hyperphosphorylated, ubiquitylated E1--E4-keratin structures was found to result in an impairment of proteasomal function. These observations shed new light on the mechanism of keratin IF network reorganisation mediated by HPV16 E1--E4 and provide an insight into the depletion of keratin co-incident with E1--E4 accumulation observed in HPV-infected epithelium.

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SUMOylation modulates the function of Aurora-B kinase.

Publication Date: 2010 Aug 15 PMID: 20663916
Authors: Fernandez-Miranda, G. - de Castro, I. P. - Carmena, M. - Aguirre-Portoles, C. - Ruchaud, S. - Fant, X. - Montoya, G. - Earnshaw, W. C. - Malumbres, M.
Journal: J Cell Sci

Aurora kinases are central regulators of mitotic-spindle assembly, chromosome segregation and cytokinesis. Aurora B is a member of the chromosomal passenger complex (CPC) with crucial functions in regulation of the attachment of kinetochores to microtubules and in cytokinesis. We report here that Aurora B contains a conserved SUMO modification motif within its kinase domain. Aurora B can bind SUMO peptides in vitro when bound to the IN-box domain of its CPC partner INCENP. Mutation of Lys207 to arginine (Aurora B(K207R)) impairs the formation of conjugates of Aurora B and SUMO in vivo. Expression of the SUMO-null form of Aurora B results in abnormal chromosome segregation and cytokinesis failure and it is not able to rescue mitotic defects in Aurora-B-knockout cells. These defects are accompanied by increased levels of the CPC on chromosome arms and defective centromeric function, as detected by decreased phosphorylation of the Aurora-B substrate CENP-A. The Aurora-B(K207R) mutant does not display reduced kinase activity, suggesting that functional defects are probably a consequence of the altered localization, rather than decreased intrinsic kinase activity. These data suggest that SUMOylation of Aurora B modulates its function, possibly by mediating the extraction of CPC complexes from chromosome arms during prometaphase.

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The cyclin A centrosomal localization sequence recruits MCM5 and Orc1 to regulate centrosome reduplication.

Publication Date: 2010 Aug 15 PMID: 20663915
Authors: Ferguson, R. L. - Pascreau, G. - Maller, J. L.
Journal: J Cell Sci

Centrosomes are the major microtubule-organizing centers in animal cells and regulate formation of a bipolar mitotic spindle. Aberrant centrosome number causes chromosome mis-segregation, and has been implicated in genomic instability and tumor development. Previous studies have demonstrated a role for the DNA replication factors MCM5 and Orc1 in preventing centrosome reduplication. Cyclin A-Cdk2 localizes on centrosomes by means of a modular centrosomal localization sequence (CLS) that is distinct from that of cyclin E. Here, we show that cyclin A interacts with both MCM5 and Orc1 in a CLS-dependent but Cdk-independent manner. Although the MRAIL hydrophobic patch is contained within the cyclin A CLS, binding of both MCM5 and Orc1 to cyclin A does not require a wild-type hydrophobic patch. The same domain in MCM5 that mediates interaction with cyclin E also binds cyclin A, resulting in centrosomal localization of MCM5. Finally, unlike its function in DNA synthesis, MCM5-mediated inhibition of centrosome reduplication in S-phase-arrested CHO cells does not require binding to other MCM family members. These results suggest that cyclins E and A sequentially prevent centrosome reduplication throughout interphase by recruitment of DNA replication factors such as MCM5 and Orc1.

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Human G3BP1 interacts with beta-F1-ATPase mRNA and inhibits its translation.

Publication Date: 2010 Aug 15 PMID: 20663914
Authors: Ortega, A. D. - Willers, I. M. - Sala, S. - Cuezva, J. M.
Journal: J Cell Sci

The post-transcriptional regulation of nuclear mRNAs that encode core components of mitochondria has relevant implications in cell physiology. The mRNA that encodes the catalytic subunit of the mitochondrial H(+)-ATP synthase subunit beta (ATP5B, beta-F1-ATPase) is localized in a large ribonucleoprotein (RNP) complex (beta-F1-RNP), which is subjected to stringent translational control during development and the cell cycle, and in carcinogenesis. Because downregulation of beta-F1-ATPase is a conserved feature of most prevalent human carcinomas, we have investigated the molecular composition of the human beta-F1-RNP. By means of an improved affinity-chromatography procedure and protein sequencing we have identified nine RNA-binding proteins (RNABPs) of the beta-F1-RNP. Immunoprecipitation assays of Ras-GAP SH3 binding protein 1 (G3BP1) and fluorescent in-situ hybridization of mRNA indicate a direct interaction of the endogenous G3BP1 with mRNA of beta-F1-ATPase (beta-F1 mRNA). RNA-bridged trimolecular fluorescence complementation (TriFC) assays confirm the interaction of G3BP1 with the 3'-UTR of beta-F1 mRNA in cytoplasmic RNA-granules. Confocal and high-resolution immunoelectron-microscopy experiments suggest that the beta-F1-RNP is sorted to the periphery of mitochondria. Molecular and functional studies indicate that the interaction of G3BP1 with beta-F1 mRNA inhibits its translation at the initiation level, supporting a role for G3BP1 in the glycolytic switch that occurs in cancer.

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Drosophila CAF-1 regulates HP1-mediated epigenetic silencing and pericentric heterochromatin stability.

Publication Date: 2010 Aug 15 PMID: 20663913
Authors: Huang, H. - Yu, Z. - Zhang, S. - Liang, X. - Chen, J. - Li, C. - Ma, J. - Jiao, R.
Journal: J Cell Sci

Chromatin assembly factor 1 (CAF-1) was initially characterized as a histone deliver in the process of DNA-replication-coupled chromatin assembly in eukaryotic cells. Here, we report that CAF-1 p180, the largest subunit of Drosophila CAF-1, participates in the process of heterochromatin formation and functions to maintain pericentric heterochromatin stability. We provide evidence that Drosophila CAF-1 p180 plays a role in both classes of position effect variegation (PEV) and in the expression of heterochromatic genes. A decrease in the expression of Drosophila CAF-1 p180 leads to a decrease in both H3K9 methylation at pericentric heterochromatin regions and the recruitment of heterochromatin protein 1 (HP1) to the chromocenter of the polytene chromosomes. The artificial targeting of HP1 to a euchromatin location leads to the enrichment of Drosophila CAF-1 p180 at this ectopic heterochromatin, suggesting the mutual recruitment of HP1 and CAF-1 p180. We also show that the spreading of heterochromatin is compromised in flies that have reduced CAF-1 p180. Furthermore, reduced CAF-1 p180 causes a defect in the dynamics of heterochromatic markers in early Drosophila embryos. Together, these findings suggest that Drosophila CAF-1 p180 is an essential factor in the epigenetic control of heterochromatin formation and/or maintenance.

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Occludin is required for cytokine-induced regulation of tight junction barriers.

Publication Date: 2010 Aug 15 PMID: 20663912
Authors: Van Itallie, C. M. - Fanning, A. S. - Holmes, J. - Anderson, J. M.
Journal: J Cell Sci

The function of occludin remains elusive. Proposed roles include maintenance of tight junction barriers, signaling and junction remodeling. To investigate a potential role in mediating cytokine-induced changes in barrier properties, we measured barrier responses to interferon-gamma plus TNFalpha in control, occludin-overexpressing and occludin knockdown MDCK II monolayers. MDCK cells show a complex response to cytokines characterized by a simultaneous increase in the transepithelial electrical resistance and a decrease in the barrier for large solutes. We observed that overexpression of occludin increased and occludin knockdown decreased sensitivity to cytokines as assessed by both these parameters. It is known that caveolin-1 interacts with occludin and is implicated in several models of cytokine-dependent barrier disruption; we found that occludin knockdown altered the subcellular distribution of caveolin-1 and that partitioning of caveolin into detergent-insoluble lipid rafts was influenced by changing occludin levels. Knockdown of caveolin decreased the cytokine-induced flux increase, whereas the increase in the electrical barrier was unaltered; the effect of double knockdown of occludin and caveolin was similar to that of occludin single knockdown, consistent with the possibility that they function in the same pathway. These results demonstrate that occludin is required for cells to transduce cytokine-mediated signals that either increase the electrical barrier or decrease the large solute barrier, possibly by coordinating the functions of caveolin-1.

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Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages.

Publication Date: 2010 Aug 15 PMID: 20647375
Authors: Higuchi, Y. - Shiraki, N. - Yamane, K. - Qin, Z. - Mochitate, K. - Araki, K. - Senokuchi, T. - Yamagata, K. - Hara, M. - Kume, K. - Kume, S.
Journal: J Cell Sci

We previously reported that embryonic stem (ES) cells cultured on M15 cells, a mesoderm-derived supportive cell line, were efficiently differentiated towards an endodermal fate, finally adopting the specific lineages of various digestive organs such as the pancreas and liver. We show here that the endoderm-inducing activity of M15 cells is in part mediated through the extracellular matrices, and that laminin alpha5 is one of the crucial components. In an attempt to establish a feeder-free ES-cell procedure for pancreatic differentiation, we used a synthesized basement membrane (sBM) substratum using an HEK293 cell line stably expressing laminin-511. On the sBM, mouse ES or induced pluripotent stem (iPS) cells sequentially differentiated into the definitive endoderm, pancreatic progenitor cells, and then insulin-expressing pancreatic beta-cells in vitro. Knockdown of ES cells with integrin beta1 (Itgb1) reduces differentiation towards pancreatic cells. Heparan sulfate proteoglycan 2 (HSPG2) knockdown and heparitinase treatment synergistically decreased the number of Pdx1-expressing cells. These findings indicate that components of the basement membrane have an important role in the differentiation of definitive endoderm lineages. This novel procedure will be useful for the study of pancreatic differentiation of ES or iPS cells and the generation of potential sources of surrogate cells for regenerative medicine.

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Muscle wasted: a novel component of the Drosophila histone locus body required for muscle integrity.

Publication Date: 2010 Aug 15 PMID: 20647374
Authors: Bulchand, S. - Menon, S. D. - George, S. E. - Chia, W.
Journal: J Cell Sci

Skeletal muscles arise by cellular differentiation and regulated gene expression. Terminal differentiation programmes such as muscle growth, extension and attachment to the epidermis, lead to maturation of the muscles. These events require changes in chromatin organization as genes are differentially regulated. Here, we identify and characterise muscle wasted (mute), a novel component of the Drosophila histone locus body (HLB). We demonstrate that a mutation in mute leads to severe loss of muscle mass and an increase in levels of normal histone transcripts. Importantly, Drosophila Myocyte enhancer factor 2 (Mef2), a central myogenic differentiation factor, and how, an RNA binding protein required for muscle and tendon cell differentiation, are downregulated. Mef2 targets are, in turn, misregulated. Notably, the degenerating muscles in mute mutants show aberrant localisation of heterochromatin protein 1 (HP1). We further show a genetic interaction between mute and the Stem-loop binding protein (Slbp) and a loss of muscle striations in Lsm11 mutants. These data demonstrate a novel role of HLB components and histone processing factors in the maintenance of muscle integrity. We speculate that mute regulates terminal muscle differentiation possibly through heterochromatic reorganisation.

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Facilitated transport and diffusion take distinct spatial routes through the nuclear pore complex.

Publication Date: 2010 Aug 15 PMID: 20647373
Authors: Fiserova, J. - Richards, S. A. - Wente, S. R. - Goldberg, M. W.
Journal: J Cell Sci

Transport across the nuclear envelope is regulated by nuclear pore complexes (NPCs). Much is understood about the factors that shuttle and control the movement of cargos through the NPC, but less has been resolved about the translocation process itself. Various models predict how cargos move through the channel; however, direct observation of the process is missing. Therefore, we have developed methods to accurately determine cargo positions within the NPC. Cargos were instantly trapped in transit by high-pressure freezing, optimally preserved by low-temperature fixation and then localized by immunoelectron microscopy. A statistical modelling approach was used to identify cargo distribution. We found import cargos localized surprisingly close to the edge of the channel, whereas mRNA export factors were at the very centre of the NPC. On the other hand, diffusion of GFP was randomly distributed. Thus, we suggest that spatially distinguished pathways exist within the NPC. Deletion of specific FG domains of particular NPC proteins resulted in collapse of the peripheral localization and transport defects specific to a certain karyopherin pathway. This further confirms that constraints on the route of travel are biochemical rather than structural and that the peripheral route of travel is essential for facilitated import.

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Tao-1 is a negative regulator of microtubule plus-end growth.

Publication Date: 2010 Aug 15 PMID: 20647372
Authors: Liu, T. - Rohn, J. L. - Picone, R. - Kunda, P. - Baum, B.
Journal: J Cell Sci

Microtubule dynamics are dominated by events at microtubule plus ends as they switch between discrete phases of growth and shrinkage. Through their ability to generate force and direct polar cell transport, microtubules help to organise global cell shape and polarity. Conversely, because plus-end binding proteins render the dynamic instability of individual microtubules sensitive to the local intracellular environment, cyto-architecture also affects the overall distribution of microtubules. Despite the importance of plus-end regulation for understanding microtubule cytoskeletal organisation and dynamics, little is known about the signalling mechanisms that trigger changes in their behaviour in space and time. Here, we identify a microtubule-associated kinase, Drosophila Tao-1, as an important regulator of microtubule stability, plus-end dynamics and cell shape. Active Tao-1 kinase leads to the destabilisation of microtubules. Conversely, when Tao-1 function is compromised, rates of cortical-induced microtubule catastrophe are reduced and microtubules contacting the actin cortex continue to elongate, leading to the formation of long microtubule-based protrusions. These data reveal a role for Tao-1 in controlling the dynamic interplay between microtubule plus ends and the actin cortex in the regulation of cell form.

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Pex11pbeta-mediated growth and division of mammalian peroxisomes follows a maturation pathway.

Publication Date: 2010 Aug 15 PMID: 20647371
Authors: Delille, H. K. - Agricola, B. - Guimaraes, S. C. - Borta, H. - Luers, G. H. - Fransen, M. - Schrader, M.
Journal: J Cell Sci

Peroxisomes are ubiquitous subcellular organelles, which multiply by growth and division but can also form de novo via the endoplasmic reticulum. Growth and division of peroxisomes in mammalian cells involves elongation, membrane constriction and final fission. Dynamin-like protein (DLP1/Drp1) and its membrane adaptor Fis1 function in the later stages of peroxisome division, whereas the membrane peroxin Pex11pbeta appears to act early in the process. We have discovered that a Pex11pbeta-YFP(m) fusion protein can be used as a specific tool to further dissect peroxisomal growth and division. Pex11pbeta-YFP(m) inhibited peroxisomal segmentation and division, but resulted in the formation of pre-peroxisomal membrane structures composed of globular domains and tubular extensions. Peroxisomal matrix and membrane proteins were targeted to distinct regions of the peroxisomal structures. Pex11pbeta-mediated membrane formation was initiated at pre-existing peroxisomes, indicating that growth and division follows a multistep maturation pathway and that formation of mammalian peroxisomes is more complex than simple division of a pre-existing organelle. The implications of these findings on the mechanisms of peroxisome formation and membrane deformation are discussed.

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Manipulating signal delivery - plasma-membrane ERK activation in aPKC-dependent migration.

Publication Date: 2010 Aug 15 PMID: 20647370
Authors: Boeckeler, K. - Rosse, C. - Howell, M. - Parker, P. J.
Journal: J Cell Sci

Members of the PKC superfamily have been implicated in various migratory models and in particular in spatially restricted processes. However, defining the precise local events that underlie the PKC-dependent processes is constrained by the unspecific nature of interventions. Here we address this problem in relation to atypical PKC (aPKC) action, which in conjunction with the exocyst complex controls the polarised delivery of promigratory signals. A drug-dependent recruitment approach was employed to manipulate the local recruitment of signals to the leading edge of migrating cells, under conditions where the aPKC-exocyst control is globally abrogated. We found that activation of ERK but not JNK at focal adhesions recovers the majority of the migratory loss attributed to ERK action, demonstrating a necessary role for active plasma membrane ERK in the downstream signalling of aPKC-dependent migration. The data further show that restored focal adhesion dynamics are a contributing mechanism through which localized ERK activity influences this aPKC-exocyst-dependent migration.

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The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus.

Publication Date: 2010 Aug 15 PMID: 20647369
Authors: Kracklauer, M. P. - Wiora, H. M. - Deery, W. J. - Chen, X. - Bolival, B. Jr - Romanowicz, D. - Simonette, R. A. - Fuller, M. T. - Fischer, J. A. - Beckingham, K. M.
Journal: J Cell Sci

Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface.

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